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Supporting InformationNear-infrared light-mediated LA-UCNPs@SiO2-C/HA@mSiO2-

DOX@NB nanocomposite for chemotherapy /PDT/ PTT and imaging

Yuhua Chen1, Feng Zhang1, Qian Wang1, Ruihan Tong1, and Huiming Lin1,2,* Fengyu Qu1,2*

1.Key Laboratory of Photochemical Biomaterials and Energy Storage Materials, Heilongjiang Province , College of Chemistry and Chemical Engineering, Harbin Normal University, Harbin, 150025, P. R. China

2. Laboratory for Photon and Electronic Bandgap Materials, Ministry of Education, Harbin Normal University, Harbin 150025, China.

Electronic Supplementary Material (ESI) for Dalton Transactions.This journal is © The Royal Society of Chemistry 2017

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Materials.

Unless specified, all of the chemicals utilized were analytical grade and utilized

without further purification. Yttrium (III) chloride hexahydrate (YCl3·6H2O),

ytterbium(III) chloride hexahydrate (YbCl3·6H2O), thulium(III) chloride hexahydrate

(TmCl3·6H2O), ammonium fluoride (NH4F), sodiun hydroxide (NaOH), methanol,

oleic acid, tetraethylorthosilicate (TEOS), CO-520, Ammonia solution (NH4OH

25%), 3-(Triethoxysilyl) propyl isocyanate (TPI), hexadecy ltrimethyl ammonium

bromide(CTAB), (3-aminopropyl) triethoxysilane (APTES), N-(3-

dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-

hydroxysuccinimide (NHS), Lactobionic acid (LA), 1.3-Diphenylisobenzofuran

(DPBF), and doxorubicin hydrochloride (DOX) were all purchased from Aladdin.

Ethanol absolute, potassium permanganate (KMnO4), magnesium sulfate anhydrous,

cychohexane, tetrahydrofuran (THF) was dried with molecular sieve and roasted

prior to use, ethyl acetate, and dimethyl sulfoxide (DMSO) were all bought from

Sinopharm Chemical Reagent Co., Ltd. Hypocrellin A (HA) was purchased from

Chengdu Biopurify Phytochemicals Ltd. 1-Octadecene was obtained from Sigma-

Aldrich. D-glucose and concentrated hydrochloric acid were purchased from Tianjin

Fengchuan Chemical Reagent Science And Technology Co., Ltd for the synthesis of

C-Dots.

Instruments.

Samples were characterized by transmission electron microscopy (TEM Hitachi H-

8100) at an accelerating voltage of 20 kV. X-ray diffraction (XRD) is characterized

using a Rigaku Ultima IV diffractometer with Cu Ka radiation (40kV, 20 mA). The

fluorescence spectra were surveyed by HORIBA FL-3. A Fourier transform infrared

(FT-IR) spectroscopy spectrometer (JASCOFT/IR-420) was used to record the

infrared spectra of these materials. Zeta potential was carried out on the Brookhaven.

Ultraviolet-visible (UV-Vis) spectra were taken on (SHIMADZU UV2550

spectrophotometer). The isotherms of N2 adsorption/desorption were measured at the

temperature of liquid nitrogen using a Micromeritics ASAP 2010M system. The pore

size distributions were calculated from the adsorption branches of the N2 adsorption

isotherms using the Barrett-Joyner-Halenda model.

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Scheme S1. Synthetic route for the NB linker.

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The Synthesis of 2-nitro-1,3-benzenedicarboxylic acid

1,3-dimethyl-2-nitrobenzene (3.95 g, 0.105 mol), water (200 mL) and sodium

hydroxide (1.6 g, 0.16 mol) were added into a three neck flask, which was stirred and

heated to 95 ºC in oil bath, then KMnO4 (16.5 g, 0.418 mol) was added in batches

over a period of 3 h. The resulting mixture was refluxed for another 20 h, cooled and

filtered, the filtrate was acidified with concentrated HCl and the precipitate was

collected and dried.

Fig.S1 The 1HNMR spectrum of 2-nitro-1,3-benzenedicarboxylic acid.

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The Synthesis of 2-nitro-1,3-benzenedimethanol

A solution of 2-nitro-1,3-benzenedicarboxylic acid (1.6 g, 38 mmol) in 10 mL

anhydrous THF (dried with molecular sieve) was added to three neck flask and cooled

to 0 ºC in the presence of N2. Besides, 1.0 M borane-tetrahydrofuran (40 mL) was

added dropwise over about 1 h with constant pressure drop funnel. The reaction

mixture was allowed to warm slowly to room temperature and stirred as well as

refluxed for another 48 h. Afterwards, Methanol (8 mL) was added into the reaction

system slowly by constant pressure drop funnel, then the mixture was evaporated with

a rotary evaporator at 40 ºC. The residue was redissolved in ethyl acetate and washed

three to four times with water（100 mL). The organic layer was dried with anhydrous

MgSO4 overnight, the next day the solvent was evaporated once again with a rotary

evaporator at 40 ºC. The resulting yellow solid was further purified by silica gel

chromatography (hexane:ethyl acetate=1:1) to obtain 2-nitro-1,3-benzenedimethanol.

Fig.S2 The 1HNMR spectrum of 2-nitro-1,3-benzenedimethanol.

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Synthesis of NB-linker

In a 100 mL three-necked flask, 2-nitro-1,3-benzenedimethanol (1.7977g, Fig. S1 and

2), was dissolved in 36 mL of anhydrous THF in the presence of Ar. And then, 3-

(Triethoxysily) propyl isocyanate (3.6251g) in 20 ml anhydrous THF was added

dropwise with constant pressure drop funnel. Finally, the reaction system was

refluxed for another 12 h in oil bath. At the procedure, the reaction flask was quickly

covered with aluminum foil to avoid the sunlight.

Synthesis of C-Dots

2.4 g (1 M) of glucose was dissolved in 10 mL of triple distilled water (TDW) and then 10

mL concentrated hydrochloric acid (11.67 M) was added to it. The mixture was sonicated for

2 h. The solution was dried completely in the oven about 80–100 °C, and then the black

carbon powder was collected. About 0.8 g of carbon powder was dissolved in 350 mL of

TDW and then again sonicated for 10 min. This solution was centrifuged at 6000 rpm and the

supernatant was collected for further study.

LA attachment

LA as covalently conjugated onto amine-functionalized

UCNPs@SiO2C/HA@mSiO2-DOX@NB linker through the -COOH group by using

cross-linking reagents EDC and NHS. In this work, firstly, 100 mg UCNPs@SiO2-

C/HA@mSiO2-DOX@NB linker was dispersed in 20 mL of toluene and 50 µL of

APTES added to the solution. The solution was heated to 50 °C and stirred for 4 h

with the flowing of argon to obtain the amino-functional nanocomparticles. Secondly,

1 mmol LA was dissolved in 2 mL of DMSO followed by the addition of 1 mmol

EDC and 1 mmol NHS. The mixture was then stirred at room temperature for 15 min

to activate the carboxylic group of LA. Subsequently, 100 µL of the above solution

was added to 5 mL of methanol solution of amino-functionalized UCNPs@SiO2-

C/HA@mSiO2-DOX@NB linker (60 mg), and the mixture was stirred for 24 h at

room temperature. Excess EDC, NHS and LA were removed by repeatedly washing

with ethanol.

Detection of singlet oxygen

DPBF was employed as a chemical probe to determine singlet oxygen by measuring the

absorption via UV−Vis spectroscopy. Typically, 2 mL of ethanol solution containing DPBF

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(10 mmol L-1) was added to 2 mL of UCNPs@SiO2-C/HA solution (0.5 mg mL-1) and then

transferred into a 5 mL cuvette. The solution was irradiated by a 980 nm NIR laser (2.15 W

cm-2) for different time, and then the solution was centrifuged, and the supernatant was

collected for UV−Vis absorption.

Photothermal testing

To investigate the photothermal effect of UCNPs@SiO2-C3/HA3, 2 mL of aqueous solutions

(500 µg mL-1) were irradiated under an NIR laser (980 nm, 2.15 W cm-2) for 25 min. A digital

thermometer was used to measure the temperature during irradiation.

The modification of FITC on LA-UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB

Firstly, 15 mg of FITC and 100 µL of APTES were dissolved to 5 mL of methanol

solution, and then the mixture was stirred for 24 h at room temperature for the next

step (FITC-APTES). Secondly, 2 mL FITC-APTES solution was added into the above

LA-UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB solution and stirred for another 24 h

at room temperature. The FITC-LA-UCNPs@SiO2-DOX-C3/HA3@mSiO2@NB was

collected by centrifugation at 6000 rpm.

Apoptosis Detection by AM

To visulize the cell apoptosis, 1 × 105 HePG2 cells were seeded on a 6-well plate and cultured

overnight in a humidified incubator at 37 °C with 5% CO2. Then, cells were treated with LA-

UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB2 (500 µg mL-1) for 4 h. Then, cells were irradiated

with NIR laser light (980 nm, 1 W cm-2) for 0-20 min. Cells were incubated for another 4 h in

fresh culture medium before being collected. Finally, cells were stained with AM for analysis.

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Fig. S3 The TEM images of (A) UCNPs@SiO2, (B) UCNPs@SiO2-C1/HA1, (C) UCNPs@SiO2-C2/HA2

Fig. S4 Low-angle XRD patterns of samples

50 nm

A

50 nm

B

50 nm

C

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Fig. S5 (A) Nitrogen adsorption-desorption isotherms and (B) pore size distribution for UCNPs@SiO2-C3/HA3@mSiO2, UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB1, UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB2, and UCNPs@ SiO2-C3/HA3@mSiO2-DOX@NB3.

The porous structures of UCNPs@SiO2-C3/HA3@mSiO2 and UCNPs@SiO2-

C3/HA3@mSiO2-DOX@NBs were further studied by N2 adsorption-desorption (Fig. S11).

Mesoporous structures derived from the mSiO2 shell that are verified by the typical IV adsorption

isotherm and H4 hysteresis loop in middle relative pressure region as shown in Fig. S11A of

UCNPs@SiO2-C3/HA3@mSiO2. And the hysteresis loop at high relative pressure region implies

the void structure derived from the accumulation of these nanoparticles. Moreover, the absorption

amounts of UCNPs@SiO2-C3/HA3@mSiO2-DOX@NBs decrease remarkably due to the drug

loading and NB-linker modification. Here, NB silane coupling agent was exploited as the “gate”

to seal the drug molecules inside the mesopore, so that with increasing the NB linker, the DOX

loading efficiency aggrandizes from 5.24±0.04 % (UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB1)

to 9.73±0.05 % (UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB3) as present in Table S1. In addition,

the DOX loading and organic modification also makes the surface area and pore volume decrease

from 563 m2 g-1and 0.64 cm3 g-1 of UCNPs@SiO2-C3/HA3@mSiO2 to 130 m2 g-1 and 0.53 cm3 g-1

of UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB1, 95 m2 g-1 and 0.35 cm3 g-1 of UCNPs@SiO2-

C3/HA3@mSiO2-DOX@NB2, 59 m2 g-1 and 0.23 cm3 g-1 of UCNPs@SiO2-C3/HA3@mSiO2-

DOX@NB3, respectively (Table S1).

A B

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Table S1 The porous parameters of the samples

Fig. S6 The UV/Vis spectrum of the samples.

Sample Surface area (m2 g-1)

Pore volume (cm3 g-1)

Pore size (nm)

Loading efficiency (%)

UCNPs@SiO2-C3/HA3@mSiO2 563 0.64 2.35

UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB1 130 0.53 2.20 5.24±0.04

UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB2 95 0.35 2.10 8.60±0.03

UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB3 59 0.23 2.00 9.73±0.05

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Fig. S7 (A) The XPS spectrum of UCNPs@SiO2-C3. (B) The high resolution XPS spectra of

C1s.

The C-Dots of this complex have been characterized by X-ray photoelectron. The XPS

spectrum of UCNPs@SiO2-C3 (Fig. S7A) shows the peak at 285, 532, 103, 158 and 698

eV，which is attribute to C1s, O1s, Si2p, Y3d and F1s, respectively. Fig. S7B reveals the

high resolution XPS spectra of C1s. The peaks at 284.3, 285.4, 287.0, and 288.2 eV suggest

the C-C, C-C/C-H, C-O-C and C=O group of C-Dots.

BA

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Fig. S8 Fluorescence spectra at various excitation wavelengths. (A) Dox, (B)C-Dots, (C) HA.

A

C

B

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Fig. S9 UCL spectra of the samples under excitation.

The corresponding upconversion luminescence (UCL) spectra under NIR laser excitation

(980 nm) were recorded as shown in Fig. S9. The different intensity emission peaks at about

344, 360, 450, 475 and 808 nm are assigned to the 1I6-3F4, 1D2-3H6, 1D2-3F4, 1G4-3H6 and 3H4-3H6 transition from Tm3+, respectively. After the growth of NaYF4 shell, NaYF4:Yb,

Tm@NaYF4 exhibits enhanced fluorescence emission. And the decreased fluorescence of

UCNPs@SiO2-C3/HA3 is ascribed to the light-scattering effect on both emission and incident

light by the silica layer. Furthermore, the emission at 450-500 nm exhibits the more inhibition

owing to the blue light absorption of HA

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Fig. S10 Absorption spectra changes of DPBF treated with UCNPs@SiO2-C3/HA3 after 980 nm irradiation for different times.

DPBF was employed as a chemical probe to evaluate ROS generation by the UV−Vis

absorption spectroscopy because DPBF can react with ROS irreversibly to cause the decrease

of its characteristic absorption. The typical absorbance of DPBF at 420 nm decreases

obviously because of the efficient generation of ROS from UCNPs@SiO2-C3/HA3

illuminated by 980 nm NIR light.

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Fig. S11 (A) Release profiles of UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB2 in the dark (0-330min) and with NIR irradiation (330-660min). (B) Release profiles of UCNPs@SiO2-C3/HA3@mSiO2-DOX with (a) and without (b) NIR irradiation, and UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB2 with (c) and without (d) NIR irradiation.

To further study the NIR-triggered release behavior, the DOX release from

UCNPs@SiO2-C3/HA3@mSiO2-DOX@NB2 in the dark and then with NIR

irradiation was recorded as shown in Fig. S11A. When the solution of UCNPs@SiO2-

C3/HA3@mSiO2-DOX@NB2 was placed under dark condition for the first 330 min,

just few leakages can be detected (below 10.23±0.03 %). After that, the NIR

irradiation was introduced and the release is enhanced obviously. In order to further

testify the role of NB “gate” on the controlled release, the release behaviors of the

nanocomposite without NB “gate” (UCNPs@SiO2-C3/HA3@mSiO2-DOX) were

investigated as shown in Fig. S11B. DOX release more freely from UCNPs@SiO2-

C3/HA3@mSiO2-DOX with and without NIR irradiation, but there is not significant

difference between the release performances of them. Both of them can reach about

90.26±0.42 % DOX release after 240 min. With NB “gate”, UCNPs@SiO2-

C3/HA3@mSiO2-DOX@NB2 reveals the excellent controlled release triggered by

NIR. Based on the above investigation, it is the sensitive NB “gate” to insure the NIR-

controlled DOX release that is significant for the operable DOX chemotherapy.

A B

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Fig. S12 UV-Vis spectra of NB linker solution after treated with UV irradiation for different time. Inset:(a) before irradiation, (b) after irradiation.

The direct evidence for the UV sensitive NB linker is present in Fig. S12. Based

on the above equation, NB linker could be broken under UV irradiation, and Fig S12

shows the UV-Vis spectra of NB linker (25 µL, 0.20 M) dissolved in PH 6.5 PBS (3

mL) treated by UV irradiation for different time. The increased absorbance at about

300~350 nm owing to the 2-nitro-1,3-benzenedialdehyde fall off from NB linker.

And the inset photo exhibits the color change for NB linker solution before and after

the UV irradiation.

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Fig.S13 DCF fluorescence images in HePG2 cells treated with LA-UCNPs@SiO2-C3/HA3 under NIR irradiation for (A) 0 min, (B) 5 min, (C) 10 min, and (D) 20 min. scale bar: (100 µm).

Intrcellular ROS generation was detected using DCFH-DA as indicator, which is

oxidized to form a green fluorescent substance (2’7’-dichlofluorescein, DCF) inside cells (Fig.

S13). After excited by 980 nm light, HePG2 cells shows enhanced green fluorescence with

the increasing the irradiation time owing to the improved ROS amount. On the contrast,

without 980 nm irradiation there is no remarked green fluorescence (Fig. S13). Above high

intracellular ROS generation capability make LA-UCNPs@SiO2-C3/HA3 as promising

candidate as the PDT agent.

DC

A B

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Fig. S14 Fluorescence images of calcein-AM (green, live cells) costained HePG2 cells after

laser irradiation for (A) control, (B) 0 min, (C) 10 min, (D) 20 min. scale bar: (100 µm).

To further visualize cell survival treated by LA-UCNPs@SiO2-C3/HA3@mSiO2-

DOX@NB2, the HePG2 cells are stained with calcein acetoxymethyl ester (calcein-AM),

which is a typical dye for living cells. From Fig. S14, with prolonging the irradiation time, the

green fluorescence derived from AM is inhibited due to the decreased cell viability. Based on

the above investigation, the synergistic effect of chemotherapy, PTT and PDT induce the

enhanced cell cytotoxicity.

A

DC

B